This invention is related to the field of detection of diterpenoid biosynthesis, particularly to the biosynthesis of taxoid compounds such as Taxol.
The highly functionalized diterpenoid TAXOL(copyright) (paclitaxel) (Wani et al., J. Am. Chem. Soc. 93:2325-2327, 1971) is well-established as a potent chemotherapeutic agent (Holmes et al., in Taxane Anticancer Agents: Basic Science and Current Status, Georg et al., eds., pp. 31-57, American Chemical Society, Washington, D.C., 1995; Arbuck and Blaylock, in Taxol: Science and Applications, Sufffess, ed., pp. 379-415, CRC Press, Boca Raton, Fla., 1995). (Paclitaxel is the generic name for TAXOL(copyright) (paclitaxel), a registered trademark of Bristol-Myers Squibb.)
The supply of TAXOL(copyright) (paclitaxel) from the original source, the bark of the Pacific yew (Taxus brevifolia Nutt.; Taxaceae) is limited. As a result, there have been intensive efforts to develop alternate means of production, including isolation from the foliage and other renewable tissues of plantation-grown Taxus species, biosynthesis in tissue culture systems, and semisynthesis of TAXOL(copyright) (paclitaxel) and its analogs from advanced taxane diterpenoid (taxoid) metabolites that are more readily available (Cragg et al., J Nat. Prod. 56:1657-1668, 1993). Total synthesis of TAXOL(copyright) (paclitaxel), at present, is not commercially viable (Borman, Chem. Eng. News 72(7):32-34, 1994), and it is clear that in the foreseeable future the supply of TAXOL(copyright) (paclitaxel) and its synthetically useful progenitors must rely on biological methods of production, either in Taxus plants or in cell cultures derived therefrom (Sufffiess, in Taxane Anticancer Agents: Basic Science and Current Status, Georg et al., eds., American Chemical Society, Washington, D.C., 1995, pp. 1-17).
The biosynthesis of TAXOL(copyright) (paclitaxel) involves the initial cyclization of geranylgeranyl diphosphate, the universal precursor of diterpenoids (West, in Biosynthesis of Isoprenoid Compounds, Porter and Spurgeon, eds., vol. 1, pp. 375-411, Wiley and Sons, New York, N.Y, 1981), to taxa-4(5),11(12)-diene (Koepp et al., J Biol. Chem. 270:8686-8690, 1995) followed by extensive oxidative modification of this olefin (Koepp et al., J Biol. Chem. 270:8686-8690, 1995; Croteau et al., in Taxane Anticancer Agents: Basic Science and Current Status, Georg et al., eds., pp. 72-80, American Chemical Society, Washington, D.C, 1995) and elaboration of the side chains (FIG. 1) (Floss and Mocek, in Taxol: Science and Applications, Suffness, ed., pp. 191-208, CRC Press, Boca Raton, Fla., 1995).
Taxa-4(5),11(12)-diene synthase (xe2x80x9ctaxadiene synthasexe2x80x9d), the enzyme responsible for the initial cyclization of geranylgeranyl diphosphate, to delineate the taxane skeleton, has been isolated from T. brevifolia stem tissue, partially purified, and characterized (Hezari et al., Arch. Biochem. Biophys. 322:437-444, 1995).
Although taxadiene synthase resembles other plant terpenoid cyclases in general enzymatic properties (Hezari et al., Arch. Biochem. Biophys. 322:437-444, 1995), it has proved extremely difficult to purify in sufficient amounts for antibody preparation or microsequencing, thwarting this approach toward cDNA cloning.
We have cloned and sequenced the taxadiene synthase gene of Pacific yew.
One embodiment of the invention includes isolated polynucleotides comprising at least 15 consecutive nucleotides, preferably at least 20, more preferably at least 25, and most preferably at least 30 consecutive nucleotides of a native taxadiene synthase gene, e.g., the taxadiene synthase gene of Pacific yew. Such polynucleotides are useful, for example, as probes and primers for obtaining homologs of the taxadiene synthase gene of Pacific yew by, for example, contacting a nucleic acid of a taxoid-producing organism with such a probe or primer under stringent hybridization conditions to permit the probe or primer to hybridize to a taxadiene synthase gene of the organism, then isolating the taxadiene synthase gene of the organism to which the probe or primer hybridizes.
Another embodiment of the invention includes isolated polynucleotides comprising a sequence that encodes a polypeptide having taxadiene synthase biological activity. Preferably, the polypeptide-encoding sequence has at least 70%, preferably at least 80%, and more preferably at least 90% nucleotide sequence similarity with a native Pacific yew taxadiene synthase polynucleotide gene.
In preferred embodiments of such polynucleotides, the polypeptide-encoding sequence encodes a polypeptide having only conservative amino acid substitutions to the native Pacific yew taxadiene synthase polypeptide, except, in some embodiments, for amino acid substitutions at one or more of: cysteine residues 329, 650, 719, and 777; histidine residues 370, 415, 579, and 793; a DDXXD motif (SEQ ID NO:3); a DXXDD motif (SEQ ID NO:4); a conserved arginine; and a RWWK element (SEQ ID NO:5). Preferably, the encoded polypeptide has only conservative amino acid substitutions to or is completely homologous with the native Pacific yew taxadiene synthase polypeptide. In addition, the encoded polypeptide preferably lacks at least part of the transit peptide. Also included are cells, particularly plant cells, and transgenic plants that include such polynucleotides and the encoded polypeptides.
Another embodiment of the invention includes isolated polypeptides having taxadiene synthase activity, preferably having at least 70%, more preferably at least 80%, and most preferably at least 90% homology with a native taxadiene synthase polypeptide. Also included are isolated polypeptides that comprise at least 10, preferably at least 20, more preferably at least 30 consecutive amino acids of a native Pacific yew taxadiene synthase, and most preferably the mature Pacific yew taxadiene synthase polypeptide (i.e., lacking only the transit peptide).
Another embodiment of the invention includes antibodies specific for a native Pacific yew taxadiene synthase polypeptide.
Another embodiment of the invention includes methods of expressing a taxadiene synthase polypeptide in a cell, e.g., a taxoid-producing cell, by culturing a cell that includes an expressible polynucleotide encoding a taxadiene synthase polypeptide under conditions suitable for expression of the polypeptide, preferably resulting in the production of the taxoid at levels that are higher than would be expected from an otherwise similar cell that lacks the expressible polynucleotide.